The presence of nucleic acids in a sample may indicate that a source from which the sample is taken has a disease, disorder or abnormal physical state. Certain diagnostics determine whether nucleic acids are present in a sample. These diagnostics invariably require amplification of nucleic acids because of the copy number problem. In a virus or cell, for example, there is usually a single copy of a particular gene. Without amplification of specific nucleic acids of the gene, it is often difficult to detect the presence of the nucleic acids.
One approach is to increase the copy number of the specific sequence, in preference to other sequences present in the sample, using an in vitro amplification method. The "polymerase chain reaction" (PCR) is one such technique (Mullis, K. et al., Cold Spring Harbor Symp. Quant. Biol. 52:263-273 (1986), Mullis, K., et al., U.S. Pat. No. 4,683,202) to selectively increase the copy number of a DNA segment having a particular sequence. In general, PCR involves treating the sample suspected of containing a target DNA sequence with oligonucleotide primers such that a primer extension product is synthesized by a DNA-dependent DNA polymerase. The primer extension product DNA strand is separated from the template strand in the preferred embodiment using heat denaturation. Both the original template and the primer extension product then serve as templates in the next and subsequent cycles of extension, resulting approximately in the doubling of the number of target DNA sequences in the sample at the end of each cycle. Consequently, multiple cycles result in the quasi-exponential amplification of the target nucleic acid sequence. Optimal practice of the PCR requires the use of a thermocycler capable of rapid changes of temperature and of a DNA polymerase, such as Taq polymerase (Saiki et al., Science 239:487-491 (1988) and Saiki, R. et al., U.S. Pat. No. 4,683,195) that resists the denaturation caused by repeated exposure to temperatures above 90.degree. C. required to separate the DNA strands.
Another in vitro amplification method referred to as the T7RT method (Burg et al., U.S. Ser. No. 080,479, abandoned) uses an RNA polymerase in addition to a DNA polymerase (a reverse transcriptase) to increase the yield of products per cycle of amplification. The method involves the use of two primers, one of which contains a promoter for the synthesis of a double-strand DNA intermediate from an RNA product by a series of primer hybridization, primer extension and product denaturation steps. The double-stranded DNA intermediate containing a promoter derived from one of the primers which directs the synthesis of multiple copies of RNA which can be used for the synthesis of additional copies of the double-stranded DNA intermediate. Multiple cycles result in an exponential amplification. The yield of products per amplification cycle exceeds that of PCR by at least an order of magnitude, thus requiring fewer cycles to obtain the same overall level of amplification. The major drawback to the T7RT method is the inherent heat denaturation step which is necessary to separate the eDNA intermediate from the RNA product but inactivates both of the thermolabile enzymes used in the process. Consequently, fresh enzymes must be added to the reaction mixture at each cycle following the heat denaturation step.
U.S. Ser. No. 07/211,384, U.S. Pat. No. 5,409,818, of Cangene Corporation describes another amplification process known as NASBA.TM., which involves the use of two primers, one of which has a promoter, and three enzymes; an RNA polymerase, a DNA polymerase (a reverse transcriptase) and a ribonuclease (RNase H) that specifically degrades the RNA strand of an RNA-DNA hybrid. The cyclic process takes place at a relatively constant temperature throughout and without serial addition of reagents, wherein the first primer hybridizes to the RNA product, reverse transcriptase uses the RNA product as template to synthesize a DNA product by extension of the first primer, RNase H degrades the RNA of the resulting RNA-DNA hybrid, the second primer with the promoter hybridizes to the DNA product, reverse transcriptase uses the second primer as template to synthesize a double-stranded promoter by extension of the DNA product, an RNA polymerase uses the promoter and DNA product to transcribe multiple copies of the same RNA product. The unique addition of RNase H distinguishes NASBA.TM. from the T7RT process by eliminating the need for heat denaturation to separate the DNA product from its RNA template.
U.S. Pat. No. 5,130,238 of Cangene Corporation describes an enhanced nucleic acid amplification process known as enhanced NASBA.TM.. The process is similar to that described in U.S. Ser. No. 07/211,384, and U.S. Pat. No. 5,409,818, and is enhanced by addition to the reaction mixture of an alkylated sulfoxide (for example, dimethyl sulfoxide) and BSA.
U.S. Ser. No. 08/275,250 of Cangene Corporation describes a further improvement of NASBA.TM.. To overcome thermal denaturation during entry into the amplification cycle from DNA, this process of amplification uses RNA polymerase, specifically, E. coli RNA polymerase to eliminate the heating steps involved in entering the amplification cycle.
Notwithstanding these amplification processes, a need exists for improvements to the amplification process. It would be preferable if the amplification process required fewer steps and fewer manipulations by a user.
One step which would be helpful to eliminate is the addition of a promoter sequence to derived DNA to allow subsequent transcription. For example, it is known that DNA synthesis from a DNA or an RNA template requires a DNA or an RNA primer with a 3'-OH group. It is also known that normal synthesis of an RNA using T7 RNA polymerase requires a double-stranded DNA promoter immediately upstream of a template from which the RNA is transcribed. Such transcribed RNA would not contain a promoter sequence; thus one would need to add such promoter sequence to derived DNA to allow subsequent transcription. In NASBA.TM. and enhanced NASBA.TM. amplification reactions, these basic requirements are met by providing two primers, a first primer which hybridizes to the RNA product, and a second primer which has a plus-sense sequence of a T7 promoter and hybridizes to the DNA product. The first primer is extended using the RNA product as template to form the DNA product which serves as the template for transcription of the RNA product. The DNA product is extended using the second primer as template to form a double-stranded promoter for the transcription of the RNA product.
This invention improves upon NASBA.TM. and enhanced NASBA.TM. amplification reactions by reducing the number of primers required in the amplification cycle through the use of an RNA with an inverted repeat sequence at its 5'-end adjacent to a minus-sense sequence of the promoter recognized by an RNA polymerase. A eDNA copy of this RNA has an inverted repeat sequence at its 3'-end adjacent to a plus-sense sequence of a promoter. Upon removal of an RNA strand, the cDNA is capable of self-prig to form a partially double-stranded DNA stem-loop structure containing a double-stranded promoter oriented toward the apex of the stem-loop. Transcription of this DNA results in multiple copies of the same RNA with an inverted repeat sequence at its 5'-end adjacent to a minus-sense sequence of the promoter. Thus, the RNA product of the transcription encodes the minus-sense of the promoter sequence recognized by an RNA polymerase, and consequently the DNA copy of this RNA is fully functional as a template for transcription without the need for the addition of a promoter-bearing primer.
Other researchers have described the use of primers with inverted repeats or "hairpins" capable of transcription in nucleic acid amplification processes, namely, Dattagupta, N., EP 0 427 073 A2, and EP 0 427 074 A2, both published May 15, 1991. However, these hairpin primers encode plus-sense promoters that direct transcription of the target sequence without incorporating the sequence of the promoter itself into the product. Thus the processes described in these patents merely mimic the transcription phase of NASBA.TM. and enhanced NASBA.TM.. Furthermore, these processes do not provide for cycling, that is, the generation of templates from products, except in ways which require participation by a user or mechanical intervention, and therefore the extent of amplification is limited.
Essentially the same process is described in Beringer, M., et aI., WO 91/18155, published Nov. 28, 1991 except that the use of ribonuclease H is incorporated to enable cycling to occur. But, because the transcription products generated in this scheme do not carry promoter sequences, cycling still requires the presence of two primers, with the disadvantage that the first primer must encode both plus-sense and minus-sense strands of the promoter. In contrast, the first primer in NASBA.TM. and enhanced NASBA.TM. simply encode the plus-sense strand: the NASBA.TM. process provides for DNA synthesis to supply the minus strand.
Thus, a need exists for an amplification process which (1) eliminates the addition of a promoter sequence to derived DNA to allow subsequent transcription, (2) reduces the number of steps involved in the process, and (3) decreases the participation and manipulations by a user.